PROJECT SUMMARY Strong evidence across species suggests that exposure to maternal western-style diets (WD) creates a long- lasting metabolic signature on the offspring liver, its innate immune system, and the microbiota. We have found that maternal WD reprograms development of offspring macrophages (M?) toward a non-reparative, inflammatory phenotype that accelerates nonalcoholic fatty liver disease (NAFLD) risk in response to WD later in life. Our findings suggest that exposure to WD has both immediate and long-term effects on offspring hematopoiesis, M? function, pro-inflammatory liver gene expression and fibrogenesis that are not resolved despite switching to a healthy diet. How maternal WD during pregnancy or lactation imparts lasting effects on offspring whole body physiology and M? function through distinct metabolic and/or transcriptional mechanisms is unknown. Our preliminary data show that gut bacteria-derived indoles, mediators of the aryl hydrocarbon receptor (AHR)-driven immune response, are decreased in WD-fed dams and their offspring concomitant with an increase in metabolic reprogramming of myeloid cells. Moreover, dietary treatment of WD-fed pregnant dams with a novel antioxidant (pyrroloquinoline quinone [PQQ]), found in high concentrations in breast milk, decreases offspring NAFLD and liver fibrosis, attenuates inflammatory M? polarization, improves gut microbial function and activates AHR signaling. These findings suggest activating AHR may hold promise for prevention of NAFLD. The goal of this proposal is to establish the mechanisms through which changes in maternal diet impact M? remodeling to increase risk for NAFLD development in later life. We hypothesize that indole-producing bacteria control NAFLD risk through reprogramming of liver M? progenitors in a time- and AHR-dependent manner. We will target gestation (embryonic d 14.5) and lactation (post-natal d 14) as critical windows of WD exposure leading to persistent effects in adulthood (16 wks of age). In Aim 1, we will pair functional assays in isolated M? from liver- and bone marrow-derived cells with single cell analyses in the liver to investigate distinct transcriptional mechanisms by which maternal WD during pregnancy or lactation disrupts normal development and function of offspring innate immune cells to affect physiology. In Aim 2, we will use germ-free mice and myeloid-specific knockout of AHR to determine how bacterial indole metabolism and AHR signaling drive remodeling of naive M? to a pro-inflammatory phenotype. Metagenomic sequencing of dam and offspring microbiota will identify microbial functions altered by maternal WD exposure. Metabolomics in serum will identify deficient metabolites, such as indoles, which we will test in gain-of-function experiments. In Aim 3, we will determine how dietary PQQ promotes metabolism of indoles during pregnancy or lactation to preserve M? function in offspring exposed to maternal WD in vivo and in vitro. Completion of these aims will vertically advance understanding of how maternal diet patterns development of the innate immune system through the microbiome and will test a novel antioxidant countermeasure, PQQ, aimed at halting developmental programming of NAFLD.